title: Immunoblotting.
abstract: Two basic methods can be used for the identification of specific antigens by their corresponding antibodies: immunoprecipitation and immunoblotting (often referred to as Western blotting) (1,2). Each has its advantages. Immunoprecipitation is likely to permit the detection of both conformational and sequential epitopes and is equally efficient at detecting low (<50 kDa)- and high-mol-wt (>50 kDa) proteins, in contrast to immunoblotting, which is usually less efficient for the detection of high-mol-wt protems. On the other hand, immunoprecipitation is less sensitive than immunoblotting and technically less convenient (3). Blotting, i.e., the immobilization of proteins (or DNA, RNA) to the surface of a membrane, allows specific methods of detection and analysis, which are not practical for molecules in solution or trapped in a gel matrix. Immunoblotting is a rapid and sensitive assay for which, especially if amplification steps are introduced, antigens can be identified with a sensitivity in the lower picogram range. Solubilization of cell-bound protems relies on harsh denaturing treatment, and the strong forces that bind the antigen to the blotmembrane may prevent complete renaturation. With respect to the possible destruction of conformational epitopes, antibodies that recognize primary protem structures should be used.
authors.affiliation: Institute for Immunology, University of Heidelberg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Kirschfink
authors.forename: P
authors.initials: P
authors.lastname: Terness